HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mou, T.-C. Articles by Gray, D. M. Search for Related Content PubMed PubMed Citation Articles by Mou, T.-C. Articles by Gray, D. M. Social Bookmarking                   What's this?

Independent tyrosyl contributions to the CD of Ff gene 5 protein and the distinctive effects of Y41H and Y41F mutants on protein–protein cooperative interactions

¹ Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 75083-0688, USA
² Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523, USA
³ Biosciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA

Reprint requests to: Donald M. Gray, Department of Molecular and Cell Biology, Mail Stop FO31, The University of Texas at Dallas, P.O. Box 830688, Richardson, TX 75083-0688; e-mail: dongray{at}utdallas.edu; fax: (972) 883-2409.

The gene 5 protein (g5p) of the Ff virus contains five Tyr, individual mutants of which have now all been characterized by CD spectroscopy. The protein has a dominant tyrosyl 229-nm L_a CD band that is shown to be approximately the sum of the five individual Tyr contributions. Tyr41 is particularly important in contributing to the high cooperativity with which the g5p binds to ssDNA, and Y41F and Y41H mutants are known to differ in dimer–dimer packing interactions in crystal structures. We compared the solution structures and binding properties of the Y41F and Y41H mutants using CD spectroscopy. Secondary structures of the mutants were similar by CD analyses and close to those derived from the crystal structures. However, there were significant differences in the binding properties of the two mutant proteins. The Y41H protein had an especially low binding affinity and perturbed the spectrum of poly[d(A)] in 2 mM Na⁺ much less than did Y41F and the wild-type gene 5 proteins. Moreover, a change in the Tyr 229 nm band, assigned to the perturbation of Tyr34 at the dimer–dimer interface, was absent in titrations with the Y41H mutant under low salt conditions. In contrast, titrations with the Y41H mutant in 50 mM Na⁺ exhibited typical CD changes of both the nucleic acid and the Tyr 229-nm band. Thus, protein–protein and g5p–ssDNA interactions appeared to be mutually influenced by ionic strength, indicative of correlated changes in the ssDNA binding and cooperativity loops of the protein or of indirect structural constraints.

Keywords: Circular dichroism; Ff gene 5 protein; tyrosine CD bands; protein–protein cooperative interactions; secondary structure analysis

Abbreviations: CD, circular dichroism • DSSP, Define Secondary Structures of Proteins computer program (Kabsch and Sander, 1983) • , L - R (or molar CD) • Ff, filamentous phages (M13, fd, and f1) that require F pili to infect E. coli • g5p, gene 5 protein • K, the intrinsic binding constant K times a cooperativity factor • n, the number of nucleotides bound per g5p monomer • OB fold, oligonucleotide/oligosaccharide binding fold • PDB, Protein Data Bank • [P]/[N], [protein monomer]/[nucleotide] molar ratio • SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis • ssDNA, single-stranded DNA • WT, wild-type protein

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?